Immunoassay techniques have been known for the last few decades and are now commonly used in medicine for a wide variety of diagnostic purposes to detect target analytes in a biological sample. Immunoassays exploit the highly specific binding of an antibody to its corresponding antigen, wherein the antigen is the target analyte. Typically, quantification of either the antibody or antigen is achieved through some form of labeling such as radio- or fluorescence-labeling. Sandwich immunoassays involve binding the target analyte in the sample to the antibody site (which is frequently bound to a solid support), binding labeled antibody to the captured analyte, and then measuring the amount of bound labeled antibody, wherein the label generates a signal proportional to the concentration of the target analyte inasmuch as labeled antibody does not bind unless the analyte is present in the sample.
Immunoassay methods can be carried out in any of a wide variety of formats. A typical heterogeneous sandwich immunoassay employs a solid phase as a support to which is bound a first (capture) antibody reactive with at least one epitope on the target analyte. A second (detection) antibody is also reactive with at least one epitope the target analyte, and may be conjugated to a detectable label that provides a signal that is measured after the detection antibody binds to the captured target analyte. The solid phase is made of a material with sufficient surface affinity to bind an antibody and can take many different forms, including a magnetic or paramagnetic microparticle composed of a suitable polymer. These magnetic or paramagnetic microparticles are used to facilitate manipulation of the microparticle within a magnetic field, so that they can be separated from a mixture of soluble reagents and a test sample using the magnetic field.
Whole blood samples are often prepared for assay by centrifuging the sample, resulting in the formation of three layers: a clear fluid layer (plasma at the top), a red fluid layer at the bottom that contains most of the erythrocytes (red blood cells), and a thin dividing buffy coat layer between the plasma layer and erythrocyte layer, which contains most of the leukocytes (white blood cells) and platelets. When centrifuging is insufficient, the layers are not completely separated and, depending on the extent and quality of centrifuging, the plasma layer may contain substantial amounts of leukocytes and platelets. Magnetic- and paramagnetic-particle based assays targeting analytes in blood serum or plasma are subject to interference from leukocytes that may remain in the plasma layer due to incomplete separation of the layers. For example, assays can provide lower than expected values when test samples are not sufficiently centrifuged in preparation for the assay, due to interference from leukocytes remaining in the plasma layer. The interference problem increases the risk of false negative diagnostic results and the risk that individuals will not obtain a timely diagnosis.
One approach to the interference problem involves adding poly-L-Lysine directly to the assay system, notwithstanding the recognition that doing so may interfere with the system by aggregating other reagents and binding members in addition to the red blood cells. The addition of poly-L-lysine is thus not always effective, particularly when a poly-anion material is used in the reaction solution. Moreover, poly-L-Lysine is costly.
Improved immunoassay methods and kits are needed, which compensate for interference from various substances such as leukocytes that may also be present in a test sample, and in particular for such methods that do so at minimal cost and without contributing another source of interference to the assay system.